PROJECT SUMMARY While malaria deaths in Africa due to Plasmodium falciparum have fallen over the last decade, the proportion of malaria cases due to non-falciparum species is rising. Improved diagnostics are needed to respond to this changing epidemiology. Yet current ?test and treat? strategies in Africa rely upon rapid diagnostic tests (RDTs) that do not reliably detect most non-falciparum infections and may be compromised by histidine-rich protein 2- (PfHRP2-) negative parasites. We propose to develop and compare novel recombinase polymerase amplification- (RPA-) and CRISPR-based diagnostic platforms that have the potential to launch the next generation of RDTs, capable of sensitive multiplexed detection of all Plasmodium species. We have already successfully adapted RPA and a new technology called SHERLOCK (Specific High Sensitivity Enzymatic Reporter Unlocking) for malaria, reaching limits of detection on par with real-time PCR. We now propose to tackle the more difficult detection of Plasmodium malariae and Plasmodium ovale. Our experience in diagnostic development and evaluation, expertise in non-falciparum and low-density malaria infections, and collaborative relationship with multiple in-country African investigators make us uniquely suited to pioneer this new technology. In Aim 1, we will develop and optimize P. malariae and P. ovale RPA and SHERLOCK assays with enhanced sensitivity and robustness, utilizing a range of strategies that have previously allowed streamlined, multiplexed detection of multiple pathogens. We will detect target nucleic acids using a multiplexed lateral flow device for RPA and simple fluorimeter for SHERLOCK. Sensitivity and specificity of the new assays will be benchmarked using a large sample bank of archived isolates from the Democratic Republic of the Congo and Tanzania. In Aim 2, we will field test the best performing RPA or SHERLOCK assays in Bagamoyo, Tanzania. We will determine their sensitivity and compare their performance to current RDTs, as well as real-time PCR. These studies will leverage promising new technology for pathogen detection to provide a low-cost platform for the study of non-falciparum malaria in low-resource settings. If successful, they will lay the groundwork for development of species-specific, sensitive RDTs capable of multiplexed detection of both P. falciparum and non-falciparum malaria in Africa.